PROJECT SUMMARY Understanding the effects of guidance cues and their receptors on the growth cone during outgrowth will be critical to a comprehensive understanding of axon guidance in normal and diseased states. The Netrin family of secreted guidance cues act as both attractants and repellants in axon guidance. While much has been learned about Netrin signaling in attractive axon guidance, Netrin-mediated repulsion is incompletely understood. Directed growth cone migration involves asymmetric protrusion of growth cone lamellipodia and filopodia toward the target of migration. The subject of this proposal is the mechanism by which guidance ligands and receptors control polarized growth cone migration. Our results indicate that Netrin signaling polarizes protrusion and F-actin in growth cones, resulting in dorsal protrusion away from the ventral Netrin source. Using live growth cone time-lapse imaging in C. elegans, we discovered that the attractive Netrin receptor UNC-40/DCC stimulates protrusion, and the repulsive Netrin receptor UNC-5/UNC-40 heterodimer inhibits protrusion. A novel signaling pathway that mediates inhibition of protrusion driven by Netrin involves the Rac GTPases MIG-2 and CED-10, the Rac GEF UNC-73/Trio, and the microtubule-interacting protein UNC-33/CRMP. CRMP (collapsin response mediating protein) has been previously implicated in semaphorin- mediated growth cone collapse, but our results show a connection to Netrin-mediated growth cone repulsion. We hypothesize that asymmetric activities of pro- and anti-protrusive forces in the same growth cone mediate polarized migration. However, we recently discovered a role in VD/DD axon guidance for a novel UNC-5B short isoform lacking most of the cytoplasmic domains. This isoform is conserved in human Unc5A. Therefore, an alternative hypothesis is that UNC-5B short isoform regulates polarity of growth cone protrusion, while the full-length isoform regulates extent of protrusion, resulting in directed growth cone migration. This proposal is to test these alternate but not mutually exclusive hypotheses about how Netrin signaling results in asymmetric growth cone protrusion and directed outgrowth.